ABSTRACT

Trichomonas vaginalis colonizes the human urogenital tract and causes trichomoniasis, the most common nonviral sexually transmitted disease. Currently, 5-nitroimidazoles are the only recommended drugs for treating trichomoniasis. However, increased resistance of the parasite to 5-nitroimidazoles has emerged as a highly problematic public health issue. Hence, it is essential to identify alternative chemotherapeutic agents against refractory trichomoniasis. Tetracycline (TET) is a broad-spectrum antibiotic with activity against several protozoan parasites, but the mode of action of TET in parasites remains poorly understood. The in vitro effect of TET on the growth of T. vaginalis was examined, and the mode of cell death was verified by various apoptosis-related assays. Next-generation sequencing-based RNA sequencing (RNA-seq) was employed to elucidate the transcriptome of T. vaginalis in response to TET. We show that TET has a cytotoxic effect on both metronidazole (MTZ)-sensitive and -resistant T. vaginalis isolates, inducing some features resembling apoptosis. RNA-seq data reveal that TET significantly alters the transcriptome via activation of specific pathways, such as aminoacyl-tRNA synthetases and carbohydrate metabolism. Functional analyses demonstrate that TET disrupts the hydrogenosomal membrane potential and antioxidant system, which concomitantly elicits a metabolic shift toward glycolysis, suggesting that the hydrogenosomal function is impaired and triggers cell death. Collectively, we provide in vitro evidence that TET is a potential alternative therapeutic choice for treating MTZ-resistant T. vaginalis. The in-depth transcriptomic signatures in T. vaginalis upon TET treatment presented here will shed light on the signaling pathways linking to cell death in amitochondriate organisms.

INTRODUCTION

Trichomoniasis is the most common nonviral sexually transmitted infection (STI) and is caused by the parasitic protozoan Trichomonas vaginalis, with more than 275 million cases reported annually worldwide (1). Infected women develop vaginitis, urethritis, and cervicitis, potentially leading to serious health outcomes, such as infertility, preterm delivery, low-birth-weight infants, susceptibility to herpesvirus and human papillomavirus infection, and cervical cancer (2). Trichomoniasis has also been considered a cofactor of human immunodeficiency virus transmission and lethal prostate cancer (3, 4). Currently, metronidazole (MTZ) and other 5-nitroimidazoles are the only recommended drugs for the treatment of trichomoniasis. However, it is estimated that approximately 5 to 10% of all clinical cases of trichomoniasis display resistance to the above-mentioned drugs (5, 6). The only option for treating MTZ-refractory trichomoniasis is to increase the dose of MTZ. However, the teratogenic effect of MTZ on animal models is well documented (7–9), and up to 12% of patients suffer from nausea (10). Hence, it is essential to identify alternative chemotherapeutic agents to combat MTZ-resistant T. vaginalis.

Tetracyclines (TETs) are broad-spectrum antibiotics that prevent the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site and thus inhibit bacterial protein synthesis (11). Additionally, TETs have been reported to possess antiparasitic activities against several protozoans, including Plasmodium falciparum, Toxoplasma gondii, Leishmania major, Entamoeba histolytica, T. vaginalis, and Giardia lamblia (12–14). However, the mode of action of TETs against these protists remains largely unknown. Previous studies indicate that the mitochondrion and the apicoplast are the likely targets of TETs in P. falciparum (15, 16), but the targets of TETs in parasites without mitochondria, such as the hydrogenosome-containing T. vaginalis, is yet to be determined.

Although expressed sequence tags (ESTs) and microarray studies have provided useful data sets for the interrogation of T. vaginalis biology (17, 18), more in-depth analyses are needed to dissect the expression of duplicated genes of this parasite. The recent development of next-generation sequencing (NGS)-based RNA sequencing (RNA-seq) has begun to revolutionize transcriptomic studies in T. vaginalis (19, 20). These studies provide gene expression data for more than 30,000 protein-coding genes and identify specific gene families and biological pathways of the parasite in response to environmental cues. As the molecular mechanisms leading to cell death in the amitochondriate organisms are poorly understood, we aim to unveil the transcriptional regulation of T. vaginalis in response to lethal stimuli using comprehensive NGS approaches. In this study, we demonstrate that TET induces cell death in both MTZ-sensitive and -resistant T. vaginalis strains. To gain insights into the antitrichomonal mechanisms of TET, we profile the most significant changes in gene expression in T. vaginalis by RNA-seq. Our results reveal that TET dramatically alters the transcriptome of T. vaginalis via activating several specific pathways and gene families, providing new insights into the molecular events governing TET-induced cell death.

Pyruvate, lactate, and acetate assays.To be consistent with the conditions used for RNA-seq, all assays were performed with the MTZ-sensitive strain unless otherwise specified. The level of intracellular pyruvate was determined with the pyruvate assay kit (BioVision). Briefly, trophozoites (2 × 106 cells) treated with TET (700 μg/ml) for 4 h were harvested and homogenized in 100 μl assay buffer. Samples (50 μl) were added to wells of a 96-well microplate. Fifty-microliter reaction mixtures (46 μl pyruvate assay buffer, 2 μl pyruvate probe, and 2 μl pyruvate enzyme mix) were added to the wells. The reaction mixture was incubated for 30 min at room temperature, and the amount of pyruvate was determined by measuring the optical density (OD) at 570 nm. The amounts of lactate and acetate in TET-treated and untreated cultures were measured with the lactate and acetate colorimetric assay kits, respectively (BioVision). The samples were prepared as for the pyruvate assay mentioned above. Reaction mixtures for the lactate assay (46 μl lactate assay buffer, 2 μl lactate probe, and 2 μl lactate enzyme mix) and for the acetate assay (42 μl acetate assay buffer, 2 μl acetate enzyme mix, 2 μl acetate substrate mix, 2 μl ATP, and 2 μl probe) were added to all wells, followed by incubation for 30 min at room temperature. The amounts of lactate and acetate were determined by measuring the OD at 450 nm.

Determination of intracellular ATP level.The intracellular ATP was determined with the ATP colorimetric/fluorometric assay kit (BioVision). Briefly, the samples were prepared as for the pyruvate assay mentioned above. Fifty-microliter reaction mixtures (44 μl ATP assay buffer, 2 μl ATP probe, 2 μl converter, and 2 μl developer mix) were added to all wells, and the reaction mixture was incubated for 30 min at room temperature. The amount of ATP was determined by measuring the OD at 570 nm.

RNA extraction, cDNA synthesis, and qPCR.Total RNA was extracted from cells treated with TET (700 μg/ml) or sterile distilled water (SDW) (control) for 4 h using the TRI reagent (Molecular Research Center). Reverse transcription (RT) was carried out using the ThermoScriptIII RT-PCR System (Invitrogen). Quantitative PCR (qPCR) was performed as previously described (17). Primer pairs used in this study are listed in Table S1 in the supplemental material.

RNA-seq library preparation and data analysis.cDNA libraries for RNA-seq were prepared as previously described (20). Briefly, total RNA from cultures treated with TET (700 μg/ml) for 4 h, which is a critical point in monitoring cell death-related events (Fig. 1A), and from cells treated with SDW (control) were isolated for mRNA purification and cDNA library construction (22). Reference transcript sequences and their annotation were downloaded from TrichDB V1.3 (23). Paired-end gene reads generated by the Illumina HiSeq sequencing platform were mapped to the T. vaginalis G3 annotated reference transcripts using CLC Genomics Workbench software (20). The mapping results are summarized in Table S2 in the supplemental material. The expression of each gene was normalized as the number of reads per kilobase per million mapped reads (RPKM value). RPKM values for all expressed genes from the TET-treated and untreated data sets are listed in Data Set S1 in the supplemental material. Differential gene expression analysis of the TET-treated transcriptome was performed using gene set enrichment analysis (GSEA) (24) and FastAnnotator (25). T. vaginalis-specific databases with functional annotation of all identified genes in the genome according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (26) were incorporated into the GSEA software as a reference database.

TET induces cell death in both MTZ-sensitive and -resistant T. vaginalis strains. The effect of TET on the growth of T. vaginalis was examined. The initial concentration of trophozoites was ∼1 × 106 cells/ml. MTZ-sensitive (ATCC 30236) (A) and MTZ-resistant (ATCC 50143) (B) strains were treated with different doses of TET (300, 500, and 700 μg/ml) and compared with the SDW-treated control. The number of viable cells was monitored every 4 h by the trypan blue exclusion assay. Data are presented as fold change in viability (mean ± SEM) of TET-treated and untreated cells (4 and 8 h) relative to 0 h from three independent experiments. Asterisks indicate statistically significant differences between the compared groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

DNA isolation and DNA fragmentation assay.Trophozoites from the TET-treated and untreated cultures were harvested and resuspended in 1 ml DNA extraction buffer (10 mM Tris-HCl, 100 mM EDTA, 30 μg/ml pancreatic RNase), followed by addition of 50 μl SDS (10%). The mixture was incubated for 1 h at 50°C, followed by addition of proteinase K (200 μg/ml) for an additional 1 h of incubation. The mixture was extracted with an equal volume of phenol (pH 8.0), and the DNA in the aqueous phase was precipitated with cold ethanol (99%). The DNA was separated on 1% agarose gels and visualized after staining by ethidium bromide (EtBr).

Nuclear staining.To detect morphological changes in nuclei, cells were stained with Hoechst 33342 (Enzol) (1:500 dilution) for 15 min. After incubation, 15 μl of the cell suspension was applied to a POC-R chamber (Zeiss) and overlaid with a coverslip. Fluorescent images were obtained using a confocal microscope (Zeiss LSM510). Excitation and emission spectra for Hoechst 33342 were examined at 350/461 nm.

Annexin V-FITC apoptosis assay.The extent of apoptosis was determined with the annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Sigma) according to the manufacturer's instructions. Briefly, approximately 1 × 106 TET-treated cells and untreated cells were prepared, washed twice with phosphate-buffered saline (PBS), and concentrated in 1 ml 1× binding buffer. Five microliters of annexin V-FITC conjugate and 10 μl of propidium iodide (PI) solution were added to each sample (500 μl), followed by incubation at room temperature for 10 min. The intensities of green fluorescence (annexin V-FITC) and red fluorescence (PI) in cells were analyzed with a flow cytometer (BD Biosciences).

Measurement of ΔΨm.The hydrogenosomal membrane potential (ΔΨm) was measured as previously described (27). Briefly, approximately 1 × 106 cells from the TET-treated and untreated cultures (3 h) were harvested and resuspended in 1 ml PBS. All tested samples, including a positive control (cells treated with 50 μM carbonyl cyanide 3-chlorophenyl hydrazone [CCCP] for depolarization of ΔΨm) were stained with 2 μM JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) (Invitrogen) and incubated at 37°C for 30 min. JC-1 can aggregate in the mitochondria as well as in the hydrogenosome (J aggregates), resulting in red fluorescence (excitation/emission = 585/590 nm) that is proportional to ΔΨm. After being washed, cells were resuspended in 0.5 ml PBS, and the changes in ΔΨm were analyzed using a flow cytometer (BD Biosciences).

Quantitation of ROS.Detection of reactive oxygen species (ROS) was performed as previously described (27). Briefly, 5 × 106 cells from the TET-treated and untreated cultures (3 h) were collected. After being washed, the cell pellets were resuspended in 1 ml PBS. A final concentration of 1 μM 2′,7′-dichlorofluorescein diacetate (2′,7′-DCF-DA) (Invitrogen) was added to each cell suspension, followed by incubation at 37°C for 1 h. The samples (100 μl) were added to a 96-well microplate, and the fluorescence was immediately detected using a fluorescence spectrophotometer (excitation/emission = 490/525 nm).

Statistical analysis.The quantitative data are presented as mean ± standard errors of the means (SEM) from three independent experiments. Student's t test (two sided) was used to test for significant differences between groups. A P value of <0.05 was considered significant.

RESULTS AND DISCUSSION

In vitro evaluation of the antitrichomonal effect of TET on the MTZ-sensitive and -resistant strains.The effect of MTZ on the growth of MTZ-sensitive (ATCC 30236) and -resistant (ATCC 50143) strains was confirmed by the viability of trophozoites in response to MTZ treatment (see Fig. S1 in the supplemental material). To determine the in vitro effect of TET on the growth of T. vaginalis, log-phase trophozoites from MTZ-sensitive and -resistant cultures were treated with different amounts of TET (300, 500, and 700 μg/ml), and the viability of the culture was monitored every 4 h. As shown in Fig. 1A, treatment with 300 μg/ml TET inhibited the proliferation of MTZ-sensitive trophozoites, with only 1.1- and 1.2-fold increments in cell number at 4 h and 8 h, respectively. Treatment with a higher concentration of TET (500 μg/ml) similarly suppressed the growth of the MTZ-sensitive strain at 4 h and 8 h. The cytotoxic effect was observed in MTZ-sensitive parasites after treatment with 700 μg/ml TET for 4 h. For the MTZ-resistant strain, treatment with 300 and 500 μg/ml TET did not inhibit the growth at 4 h (Fig. 1B), but the cytotoxic effect was observed after treatment with 500 and 700 μg/ml TET for 8 h.

A 17-fold increase in MTZ-resistant trichomoniasis in 1997 to 1998 has been reported (28). No alternative drugs are approved for treatment of the resistant cases, which remains the major challenge in controlling trichomoniasis. Several studies have evaluated in vitro the antitrichomonal activities of other compounds as potential therapeutic strategies for trichomoniasis. For example, Sapindus saponin, a component of the herbal local contraceptive Consap, has been shown to induce cell death in T. vaginalis (29). Additionally, methyl jasmonate, a small lipid derived from plants, was further demonstrated to be cytotoxic to the MTZ-resistant strain (30). Recently, resveratrol has also been proven to inhibit trichomonad growth via alteration of the hydrogenosomal metabolism (27). TET in combination with quinine has been implicated in treating patients with uncomplicated malaria (31). The use of TETs against the growth of T. vaginalis was mentioned 2 decades ago (14), reporting that the 50% inhibitory concentration (IC50) is ∼100 μg/ml. It should be noted that the maximum TET concentration was achieved at 2 h after oral administration, with approximately 1 μg/ml in human plasma (32). Hence, oral administration of TET is unlikely to be an effective treatment for trichomoniasis. Instead, due to the localized infection of T. vaginalis in the vagina, TET can be delivered by intravaginal administration, similar to the clinical use of Talsutin vaginal tablets (100 mg of tetracycline and 50 mg of amphotericin B) in the treatment of bacterial and candidal vaginitis. The usual dosages for treatment in gravid and nongravid patients are 1 and 2 tablets, respectively. Previous studies indicate that the production of vaginal fluid does not exceed 1 ml in the vagina at any time (33), suggesting that the vaginal concentration of TET after intravaginal administration is sufficient to eliminate T. vaginalis infection. Here, we show that TET potentially could serve as an alternative treatment of refractory trichomoniasis.

TET-induced cell death exhibits some apoptosis-like features in T. vaginalis.Some of the morphological features resembling apoptosis, such as chromatin condensation and reduction of nuclei size, have been identified in T. vaginalis upon treatments with proapoptotic drugs (34). To verify whether TET-induced cell death is through an apoptosis-like mechanism, we performed nuclear staining, DNA laddering, and annexin V-FITC assays to determine the type of cell death. As shown in Fig. 2A, DNA fragmentation was observed after treatment of the trophozoites with TET. The distribution of DNA in a portion of cells partially dispersed into the cytoplasm upon TET treatment (700 μg/ml), compared with the concentrated signals in the nuclei of the untreated control. The TET-treated cells exhibited a round shape, maintenance of plasma membrane integrity, and a rough cell surface (Fig. 2A). It is noteworthy that DNA laddering, a typical hallmark of apoptosis, was absent in T. vaginalis treated with TET (Fig. 2B), consistent with previous observations in T. vaginalis after treatments with antitrichomonal agents (30, 34). Moreover, using annexin V combined with PI staining, we examined phosphatidylserine (PS) exposure on the external leaflet of the plasma membrane to monitor the extent of apoptosis in T. vaginalis treated with TET. The trophozoites treated with 500 μg/ml TET had a significantly increased population of early apoptotic cells (29% ± 2.2% of cells with annexin V-FITC-positive/PI-negative signals) compared with the untreated control (14.8% ± 0.6%), whereas a higher concentration of TET (700 μg/ml) led to a shift to late apoptosis (36.6% ± 2.9% of cells with annexin V-FITC-positive/PI-positive signals) (Fig. 2C). We show here that the TET treatment results in cell death of T. vaginalis with some apoptosis-like features. Nevertheless, determination of the specific molecular machinery exerting the cell death in the amitochondriate trichomonads awaits further investigation.

TET treatment causes some apoptosis-like features in T. vaginalis. Trophozoites from TET-treated cultures (500 and 700 μg/ml for 4 h) were subjected to apoptosis-related assays and compared with untreated control (Ctrl) cells. (A) Fluorescent staining of nuclei in TET-treated and untreated cells was performed using Hoechst 33342 and examined by confocal microscopy. Scale bars, 5 μm. The right panel shows the quantitation of fragmented nuclei. The data are presented as mean ± SEM for three different groups (30 cells, 10 cells/group). (B) DNA (∼500 ng) extracted from the TET-treated and untreated cultures was separated on a 1% agarose gel and stained with ethidium bromide. M1, 100-bp marker; M2, λH3 marker. (C) Flow cytometric analysis of apoptosis in TET-treated groups compared with the control using annexin V-FITC and PI. Approximately 1 × 106 cells from the TET-treated and untreated cultures were stained with annexin V and PI to determine the apoptotic cell population. Representative results show the populations of viable (annexin V− PI−), early apoptotic (annexin V+ PI−), late apoptotic or necrotic (annexin V+ PI+), and necrotic (annexin V− PI+) cells. Mock, negative control without staining. The right panel shows the quantitation of apoptotic cells. Asterisks indicate statistically significant differences between the treated groups and the control (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Transcriptome profiling of T. vaginalis upon treatment with TET.Studies on the mode of cell death in trichomonads have focused mainly on the morphological changes in response to death stimuli (30, 34, 35), and the molecular events that occur during cell death remain unclear. To investigate the impact of TET on the transcriptome of T. vaginalis, we conducted RNA-seq analyses to identify genes with significant changes in TET-treated cells (700 μg/ml for 4 h) compared with untreated cells (SDW for 4 h). Totals of 55,206,982 and 54,943,022 raw sequencing reads were generated from the TET-treated and untreated cDNA libraries, respectively (see Table S2 in the supplemental material). Of these reads, 50,726,076 (91.88%) and 49,074,310 (89.32%) from the TET-treated and untreated data sets, respectively, were matched to the genome of T. vaginalis, confirming the quality of the cDNA libraries. The reads mapped to a total of 46,012 and 38,767 protein-coding genes expressed in the TET-treated and untreated transcriptomes, respectively (see Data Set S1 in the supplemental material). Transcripts with RPKM values of <10 under both conditions were excluded from the data sets for further differential gene expression analysis due to the low expression level (36). To validate the RNA-seq data, we determined the expression levels of 10 genes with 5-fold changes upon TET treatment by qPCR (see Table S1 in the supplemental material). qPCR analysis confirmed the expression patterns of these transcripts as observed in our RNA-seq data (see Fig. S2 in the supplemental material). Comparison of the RPKM values of transcripts between the two data sets showed a low coefficient of determination (r2 = 0.19) (see Fig. S3 in the supplemental material), suggesting that TET treatment extensively remodels the transcriptome of T. vaginalis.

GESA reveals specific pathways in response to TET.To globally assess the changes in the TET-treated transcriptome, we analyzed the differentially expressed genes by gene set enrichment analysis (GESA) (24). A total of 10,695 genes ranked by fold change in gene expression (RPKM values in the TET-treated data set compared to those in the untreated control) were subjected to GSEA, which was incorporated with the T. vaginalis-specific functional annotation databases. The enrichment data revealed that the gene sets involved in aminoacyl-tRNA biosynthesis, ribosome biogenesis, pyruvate metabolism, and glycolysis were the most upregulated cellular processes in the TET-treated transcriptome (Fig. 3A; see Table S3 in the supplemental material), whereas the gene sets associated with N-glycan biosynthesis, oxidative phosphorylation, DNA replication, and nucleotide excision repair were the most downregulated pathways (Fig. 3A; see Table S4 in the supplemental material).

Comparative transcriptomic analysis of T. vaginalis upon TET treatment. (A) A total of 10,695 transcripts ranked by fold change in gene expression (RPKM of genes in the TET-treated transcriptome [700 μg/ml for 4 h] compared with that in the untreated control) were subjected to GSEA. The enrichment plots present the most enriched upregulated or downregulated pathways in the TET-treated transcriptome according to the enrichment score (ES), which reflects the degree that a gene set is overrepresented at the top or bottom of a ranked list of genes. Positive and negative ESs indicate upregulation and downregulation of a specific gene set, respectively. Detailed information for each enriched pathway is listed in Tables S3 and S4 in the supplemental material. (B and C) GO functional classification of the most expressed genes (top 500) in the untreated transcriptome (B) compared with the TET-treated transcriptome (C). The data are presented as the percentage of each functional category among the most expressed genes.

To examine whether the differentially regulated pathways were among those that are highly expressed, we analyzed the top 500 most abundant transcripts from the RNA-seq data sets, which represented 57.2% and 56.7% of total RPKM values in the TET-treated and untreated transcriptomes, respectively (Fig. 3B and C; see Data Set S1 in the supplemental material). Gene ontology (GO)-based functional annotation of these highly expressed genes revealed that the genes encoding ribosomal proteins for the translation process represented the most abundant category in both the untreated (50%) and TET-treated (32%) transcriptomes. Interestingly, the genes encoding enzymes related to carbohydrate metabolism, including carbon utilization (9%) and glycolysis (3%), were specifically induced by TET (Fig. 3C). Also, the genes encoding several aminoacyl-tRNA synthetases (ARSs) participating in the threonine metabolic process (2%) were particularly upregulated in the TET transcriptome. Together, genes encoding ARSs and carbohydrate metabolism-related proteins were abundantly expressed, and the expression was upregulated by TET treatment, suggesting that these gene families may have potential roles during the TET-induced cell death.

ARSs are dramatically upregulated in T. vaginalis upon TET treatment.ARSs incorporate the appropriate amino acid onto its tRNA, providing the charged tRNAs for construction of peptide chains. The ARS family was the most enriched category in the TET-treated transcriptome (Fig. 3A; see Table S3 in the supplemental material). There are 34 ARSs identified in our data sets, representing 18 unique ARSs (total RPKM values for TET-treated and untreated cells were 17,899 and 811, respectively) (Fig. 4A; see Table S5 in the supplemental material). We selected 2 ARSs with significant upregulation in TET-treated cells for qPCR validation, both of which had a more than 20-fold induction compared with untreated cells (Fig. 4B).

The ARS family is dramatically upregulated in T. vaginalis in response to TET. (A) The ARS family (n = 34) is the most enriched cellular process on the basis of GSEA. The data are presented as the RPKM value of each ARS in the TET-treated transcriptome compared with the untreated transcriptome. Detailed information for each gene is provided in Table S5 in the supplemental material. (B) Validation of the RNA-seq data by qPCR. Two ARS genes (glutaminyl-tRNA synthetase [QRS] [TVAG_047990] and lysyl-tRNA synthetase [LysRS] [TVAG_267950]) with significant upregulation in the TET-treated transcriptome were selected for qPCR analysis. The data are presented as the mean ± standard deviation from three independent experiments.

In addition to functioning in protein synthesis, ARSs have been shown to possess noncanonical activities modulating cell survival (37). For example, glutaminyl-tRNA synthetase (QRS) inhibits apoptosis through the interaction with apoptosis signal-regulating kinase 1 (ASK1) in mammalian cells, which is positively regulated by its substrate glutamine (38). Recently, QRS was found to be overexpressed at the early stage of programmed cell death in E. histolytica (39). In Saccharomyces cerevisiae, the expression of glycyl-tRNA synthetase (GlyRS) GS2 is stress inducible, and the induction under stress conditions can rescue the activity of the other paralog, GS1 (40). In our RNA-seq data, the expression levels of the putative QRSs (TVAG_047990 and TVAG_258380) and GlyRS (TVAG_398680 and TVAG_398690) were drastically increased upon TET-mediated death stimuli, with 83- and 20-fold induction, respectively (see Table S5 in the supplemental material). Similarly, a recent report indicates that ARSs are highly induced in human cells treated with doxycycline, one of the TET antibiotics (41). Hence, these observations raise the possibility that overexpression of QRSs or other ARSs may be a cytoprotective response of T. vaginalis to TET, although this cannot alter the final outcome of cell death. We do not exclude the possibility that TET inhibits the protein synthesis of T. vaginalis, similar to the known mechanism in bacteria, and upregulation of ARSs may seek to compensate for the defect in protein synthesis. However, further experimental evidence is needed to confirm this hypothesis.

TET elicits a metabolic shift toward glycolysis and glycogenolysis in T. vaginalis.The main energy source for T. vaginalis comes from fermentative carbohydrate metabolism. Pyruvate produced from glucose or glycogen is subsequently metabolized in the trichomonad hydrogenosome for ATP production (42). Based on GSEA, most genes encoding glycolytic and pyruvate metabolism-related enzymes were significantly upregulated in TET-treated cells (Fig. 5A; see Table S6 in the supplemental material). The intracellular pyruvate in TET-treated and untreated cells was determined to verify this result. Obviously, a large amount of pyruvate was detected in TET-treated cells, with approximately an 8-fold induction compared with untreated cells (Fig. 5B). Pyruvate accumulation may be due to the activation of amino acid metabolism (43); however, it appears that the enzymes catalyzing the conversion of amino acids such as alanine, tryptophan, and serine to pyruvate were repressed upon TET treatment (see Fig. S4 and Table S7 in the supplemental material), which was also supported by GSEA (see Table S4 in the supplemental material). These data suggest that TET triggers a metabolic switch toward a more glycolytic phenotype. Recently, doxycycline was proven to reduce proliferation and enhance glycolytic metabolism of human cell lines (41), which is similar to our findings in T. vaginalis. Pyruvate can be converted to lactate or transported into hydrogenosome for acetate and energy production. We also found that lactate and acetate production was reduced in TET-treated cells (Fig. 5C and D), supporting massive accumulation of pyruvate.

Unexpectedly, glucokinase (GCK), which catalyzes the first step of glycolysis, was distinctly downregulated (total RPKM values for TET-treated and untreated cells were 396 and 456, respectively) (Fig. 5A; see Table S6 in the supplemental material). Downregulation of GCK can reduce the production of glucose-6-phosphate (glucose-6-P) from glucose. However, the genes encoding glycogenolytic enzymes that catalyze the reactions from glycogen to glucose-6-P were remarkably upregulated in TET-treated cells (Fig. 5A; see Table S6 in the supplemental material), which can provide a substrate for glycolysis. Both glycogen phosphorylase (TVAG_348330 and TVAG_509780) and phosphoglucomutase (TVAG_209510, TVAG_405900, TVAG_300510, TVAG_054830, TVAG_450680, and TVAG_027620) were markedly induced by TET, with 42- and 6-fold increases in gene expression, respectively. Glycogen comprises up to 15% of the dry weight of some trichomonads, implying critical roles in cell physiology (44). It is possible that elevated glycogenolysis coordinates with the downstream-activated glycolytic enzymes to enhance glycolysis in TET-treated cells. It has been reported for T. vaginalis and its related trichomonad Tritrichomonas foetus that synthesis and utilization of glycogen are responsible for abundant and deficient extracellular carbohydrate, respectively (45, 46). It remains to be determined whether the activation of glycogenolysis is due to the suppression of glucose utilization.

TET-induced cell death is not due to the acidic stress derived from glycolysis.It has been suggested that the acidic metabolites produced in S. cerevisiae under glucose-rich cultivation are the primary factor triggering an apoptosis-like response (47). We previously found that glucose-rich cultivation of T. vaginalis for 36 h reduces the pH to ∼4.2, similar to that in the vagina (48), potentially leading to cell death in T. vaginalis (20). Hence, we sought to determine whether a TET-induced glycolytic burst drives cell death in the same manner. Unexpectedly, the pH of the untreated culture was reduced from 5.75 to 5.22 within 8 h, whereas the TET-treated cultures, especially at higher concentrations (500 and 700 μg/ml), maintained the pH (5.54 to 5.61) during the treatments (see Fig. S5 in the supplemental material). It is possible that the maintenance of pH in the TET-treated cultures is partly due to less lactate and acetate production, as mentioned above. Hence, these data suggest that the cell death triggered by TET is not caused by low pH.

TET specifically inhibits the expression of NADH dehydrogenase and Fd in the hydrogenosomal energy metabolism.The genes encoding enzymes involved in the hydrogenosomal energy metabolism were mostly upregulated, except for genes encoding NADH dehydrogenase and ferredoxin (Fd) (Fig. 6; see Table S8 in the supplemental material). NADH dehydrogenase, which is composed of two subunits (TVAG_296220 and TVAG_489800 represent 24-kDa and 51-kDa subunits, respectively), has been considered a remnant of mitochondrial complex I in T. vaginalis (49). The expression levels of the two subunits were differentially regulated by TET. Of these, the 51-kDa subunit was downregulated by ∼3-fold in TET-treated cells. The Fd family genes (TVAG_003900, TVAG_399860, and TVAG_292710) were the most expressed genes in the hydrogenosome of untreated cells (2 of 3 genes with RPKM values of >2000); however, the expression was extremely downregulated by TET treatment (total RPKM values for TET-treated and untreated cells were 511.05 and 5,368.25, respectively) (see Table S8 in the supplemental material).

Pyruvate-ferredoxin oxidoreductase (PFO) catalyzes the transfer of electrons from pyruvate to Fd, which is further reoxidized by iron hydrogenase (Tvhyd) for molecular hydrogen production (43). Unlike the mitochondrial NADH dehydrogenase, the purified hydrogenosomal enzyme can also reduce Fd for molecular hydrogen production (49). It has been demonstrated that TETs can selectively inhibit the protein synthesis of complexes of the mitochondrial electron transport chain (ETC) (50), thereby leading to reduced oxidative phosphorylation and a compensatory shift toward glycolysis (41). It is likely that the reduced expression of Fd and NADH dehydrogenase interferes with the process of hydrogenosomal ETC, resulting in the imbalance of redox homeostasis and impairment of energy production in the hydrogenosome. GSEA also supported that the genes involved in the oxidative phosphorylation were negatively enriched in the TET-treated transcriptome (see Table S4 in the supplemental material). It should be noted that ATP production in the hydrogenosome is proportional to the pyruvate level (43). As the overall ATP production was not altered in TET-treated cells (Fig. 5E), it is suggested that the surplus pyruvate production was not completely metabolized due to compromised energy machinery in the hydrogenosome. Additionally, it is possible that the enhanced glycolysis can compensate for ATP generation in the cytosol. The reduced acetate production upon TET treatment could also support that the hydrogenosomal energy metabolism was impaired. A previous study indicates that Fd knockout cells show a 95% decrease in PFO enzymatic activity, a trend that is inconsistent with the mRNA level (51). Hence, it is likely that TET-induced dramatic downregulation of Fd expression may result in decreased PFO activity, which thereby could not efficiently catalyze the decarboxylation of pyruvate to produce ATP and acetate in the hydrogenosome. Together, these results suggest that the TET-induced cell death is not directly related to impairment of energy production.

TET causes hydrogenosomal dysfunction and induces ROS production in T. vaginalis.The reduced efficiency of the hydrogenosomal energy metabolism in TET-treated cells suggests that TET disrupts a hydrogenosomal function(s). Hydrogenosomal membrane potential (ΔΨm) has been shown to be an indicator of hydrogenosomal function (27, 34). To verify whether the hydrogenosome is a key target of TET, we determined the ΔΨm using the fluorescent probe JC-1 (27) in TET-treated cells compared with the untreated control. Treatment of trophozoites with CCCP, which has been shown to depolarize the mitochondrial membrane potential, indeed resulted in a significant loss of ΔΨm, as evidenced by the reduction of red fluorescence intensity (Fig. 7A to C). Notably, the ΔΨm of TET-treated cells decreased in cells treated with 500 and 700 μg/ml of TET (Fig. 7A to C), suggesting that TET destroys the hydrogenosomal activity, which may have a deleterious effect on the viability. Mitochondrial dysfunction is an early event in the process of apoptosis, which can be characterized by the changes in its morphology (52). To further determine whether TET altered the hydrogenosomal distribution of T. vaginalis, we monitored the rubrerythrin (Rbr) (TVAG_064490)-targeted hydrogenosomes (53) by immunofluorescence assay. We observed that cells treated with TET at concentrations lower than 500 μg/ml exhibited more fragmented Rbr signals than those in the untreated control (Fig. 7D). The trophozoites treated with 700 μg/ml TET displayed a diffused Rbr pattern, suggesting that the hydrogenosomal membrane integrity was interrupted. This observation was similar to previous findings in mitochondria, indicating that mitochondria often fragment into small units during apoptosis (52).

TET promotes ΔΨm dissipation, hydrogenosomal fragmentation, and ROS production in T. vaginalis. Approximately 1 × 106 cells from the TET-treated cultures (300, 500, and 700 μg/ml for 3 h) and the untreated control (Ctrl) were stained with the florescent probe JC-1 to monitor the changes in ΔΨm. (A) Representative fluorescent images of JC-1 staining in cells treated with TET compared with the control. Cells treated with CCCP, which dissipates the ΔΨm, served as a positive control for JC-1 staining. Hydrogenosomal membrane depolarization was indicated by a decrease in the red fluorescence intensity. Scale bars, 5 μm. (B) Flow cytometric analysis of the ΔΨm in the TET-treated groups compared with the control. Representative histograms show the intensity of red fluorescence in cells upon TET treatment compared with the control. (C) Quantitation of the red fluorescence intensity in the TET-treated groups compared with the untreated groups. (D) Representative fluorescent images of the hydrogenosomal morphologies in TET-treated cells compared with the control. The hydrogenosomes were stained with anti-Rbr antibody and examined by confocal microscopy. The nuclei were stained with DAPI. (E) Effect of TET on differential expression of genes involved in the antioxidant system in the hydrogenosome of T. vaginalis. The gene expression changes were determined as log2 fold changes (RPKM of genes in the TET-treated transcriptome compared with the control). Red and green indicate upregulation and downregulation by TET, respectively. (F) Determination of intracellular ROS production in TET-treated cells compared with control. TET-treated and untreated cells were stained with the probe 2′,7′-DCF-DA, and ROS production was quantified by the intensity of fluorescence. H2O2 served as a positive control. The quantitative data are presented as mean ± SEM from three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001). SOD, superoxide dismutase; TrxP, thioredoxin peroxidase; Rbr, rubrerythrin.

It has been shown that oxidative stress causes mitochondrial depolarization and fragmentation, finally leading to mitochondrial dysfunction and apoptosis (54, 55). Interestingly, we noted that the expression levels of many antioxidant-related genes identified in the T. vaginalis genome (56) were significantly repressed by TET (see Table S9 in the supplemental material). The T. vaginalis hydrogenosome contains a thioredoxin-linked peroxiredoxin antioxidant system to reduce the harmful effects of ROS (57). The main antioxidant enzymes participating in the removal of ROS in this system, such as superoxide dismutase (SOD), thioredoxin peroxidase (TrxP), and Rbr, were dramatically downregulated upon TET treatment (Fig. 7E), implying severe accumulation of ROS. To clarify whether the induction of oxidative stress was a possible cell death mechanism induced by TET, intracellular ROS production was determined in TET-treated cells using the fluorescent probe 2′,7′-DCF-DA. As expected, the trophozoites treated with hydrogen peroxide (H2O2) promoted intracellular ROS production. It is noteworthy that the levels of ROS were significantly elevated in cells treated with higher concentrations of TET (500 and 700 μg/ml) (Fig. 7F), suggesting that TET may enhance oxidative stress to activate cell death signaling. Taking the results together, we propose that TET-treated cells may have a compromised hydrogenosomal antioxidant system, resulting in ROS overproduction, which subsequently leads to dissipation of ΔΨm, hydrogenosomal fragmentation, and finally cell death.

In summary, we demonstrate, for the first time, the potential for employing TET as an alternative treatment for MTZ-resistant T. vaginalis. This new application for an old drug can significantly reduce the time and money needed to treat refractory trichomoniasis. We show that TET treatment drastically alters the transcriptome of T. vaginalis. Several molecular components and gene families that are unexpectedly enriched in T. vaginalis in response to TET-mediated cytotoxicity were also highlighted. This implies that multiple molecular events driven by the complex transcriptional regulation are involved in the process of TET-mediated cell death. Moreover, TET disrupts the antioxidant system and the ΔΨm of hydrogenosome, promoting production of ROS, which may be the downstream effectors triggering cell death in T. vaginalis. However, determination of the mechanistic details of each TET-enriched molecular event contributing to cell death requires further investigations, which will advance our understanding of cell death in amitochondriate organisms.

ACKNOWLEDGMENTS

This work was supported by grants from the Chang Gung Memorial Hospital Research Funding (CMRPD1B0441-3) and Ministry of Science and Technology, Taiwan (MOST-101-2320-B182-025-MY3), to P.T.

We thank Yi-Ywan Margaret Chen for critical review and comments on the manuscript and Chia-Jung Wu for technical assistance with flow cytometry analysis.